Comparison of Orchard-grass and Sweet Maize for Doubled Haploid Plant Production via Wide Hybridization in Bread Wheat

Turkish Journal of Agriculture - Food Science and Technology, 8(7): 1548-1552, 2020 DOI: https://doi.org/10.24925/turjaf.v8i7.1548-1552.3436

Turkish Journal of Agriculture - Food Science and Technology

Comparison of Orchard-grass and Sweet Maize for Doubled Haploid Plant

Production via Wide Hybridization in Bread Wheat

Süleyman Avcı1,a,*_{, İmren Kutlu}2,b

1_{Department of Field Crops, Faculty of Agriculture, Eskişehir Osmangazi University, 26160 Eskişehir, Turkey} 2

Department of Biosystem Engineering, Faculty of Agriculture, Eskişehir Osmangazi University, 26160 Eskişehir, Turkey

* Corresponding author A R T I C L E I N F O A B S T R A C T Research Article Received : 15/03/2020 Accepted : 19/05/2020

In this study, the potential of haploid regeneration was investigated in hybridization of six bread wheat F1 hybrids known response to another culture with orchard-grass (Dactylis glomerata L.) and sweet maize varieties (Baron, Challenger and Merit). A total of 150 wheat spikes were pollinated with orchard grass and sweet maize and 2730 pseudo-seeds were produced. Although the high rate of developed pseudo-seeds was developed from bread wheat F1 hybrids × orchard-grass, no embryos were produced. Developed pseudo-seeds (2057 number) of bread wheat × sweet maize produced 53 haploid embryos and only 8 of them were regenerated. Developed green plantlets were vernalized and applied colchicine and only four of them produced fertile seeds. The highest rate (5.9) of haploid embryo formation within wheat genotypes was determined in DH20 × Kate A-1. Although the highest haploid embryo formation was observed in Challenger with 3.5% among sweet maize genotypes, it had no effect on plant regeneration. Also, the mixture of pollen of sweet maize varieties increased haploid plant regeneration. It has been observed that some F1 hybrids such as DH20 × Kate A-1 and DH6 × Altay 2000 with low anther response gave better results in terms of haploid embryo formation and regeneration. The means of fertile spike percentages and number of seeds per fertile spike were 26.75% and 9.83, respectively in developed green plants. As a result, bread wheat × sweet maize hybridization will be a good alternative to obtain a homozygous line in a short time in bread wheat genotypes with low anther response. Keywords: F1 generation Hybridization Embryo rescue Maternal haploid Pseudo-seeds a _{savci@ogu.edu.tr}

https://orcid.org/0000-0002-4653-5567 ikutlu@ogu.edu.tr https://orcid.org/0000-0002-3505-1479

This work is licensed under Creative Commons Attribution 4.0 International License

Introduction

In wheat, which is vital for human nutrition, the limit of genetic variability has been approached to solve yield and quality problems. Accelerated breeding technologies are required for establishing wide variability in a short time, improve the efficiency of the selection, and quicken the occurring of new varieties. Double haploid technology is widely used for these aims. Producing numerous genotypes, economically, is related to use haploids in a wheat breeding program effectively. The most important advantage of using haploids is to offer the possibility of obtaining complete homozygosity.

Haploid plants in wheat can be obtained in artificial nutrient media by using anther and microspore cultures and wide hybridization of wheat with maize, cogongrass, etc. (Dang et al., 2011; Prasanna et al., 2012). Although anther and microspore culture are more widely used because they are easily applied methods, the root system

of the regenerant plant produced with a wide hybridization is healthier, and no albino plant formation is observed, which makes this method advantageous (Niu et al., 2014). Wheat × maize wide hybridization requires intensive workload; however, many researchers suggest that it is the most reliable and practical method in wheat breeding (Campell et al., 2000; Garcia-Llamas et al., 2004).

Growth conditions, pollen sources, plant growing methods, and wheat genotypes are important factors for producing doubled haploid plants, as well as the types and concentrations of the chemicals used (Knox et al., 2000). In previous studies, the effects of wheat genotype (Ltifi et al., 2019), pollen sources (Ding et al., 2019), emasculation methods (Patial et al., 2019) and hormone applications (Warchoł et al., 2016) on haploid plant regeneration of wheat were investigated.

1549 In addition to maize, Pennisetum glaucum L. (Inagaki

and Mujeeb-Kazi, 1995), sorghum (Maluszynski et al., 2003) and Imperata cylindrica (L.) Raeusch. (Chaudhary et al., 2005) species have been used to obtain haploid plants by wide hybridization with wheat. Zenkteler and Nitzsche (1984) reported that wheat, barley and rye were tested for haploid embryo formation with 15 species of Poaceae and Panicoideae and embryo formation can be obtained with

Agropyron repens (L.) P. Beauv., Alopecurus agrestis L., Dactylis glomerata L., Pennisetum americanum L. and Zea mays L. However, it is important to investigate

undiscovered pollen sources that will provide high haploid regeneration in different wheat genotypes and hybrids.

In this study, the potential of haploid plant regeneration was investigated by crossing six bread wheat F1 hybrids, response to another culture are known, with

sweet maize (Z. mays var. saccharata) and orchard-grass (D. glomerata).

Materials and Methods

In the study, six bread wheat F1 hybrids known anther

culture response were used and their features were shown in Table 1. As an inducer, the population of orchardgrass (D. glomerata) which is distributed in Campus of Agriculture Faculty, Eskişehir Osmangazi University, and sweet maize (Z. mays var saccharata) varieties [(Baron (B), Challenger (C) and Merit (M)] and mixture pollen of maize (MP) were used.

After wheat seedlings were vernalized at 4°C for 5 weeks, they were grown in pots containing soil, peat and vermiculite (3:2:1). Maize seeds were sown periodically every week at four times, and the flowering period of the wheat plant was provided to coincide with maize. Wheat spikes that slightly emerged from flag leaves were emasculated, and then these spikes were covered with isolation envelopes. Emasculated spikes were pollinated with inducer plants within 1-3 days. 2.4-D solution (213.05 mg/l, pH = 10.36) was applied within 24-48 hours following hybridization to support healthy embryo formation as described by Niu et al. (2014).

Spikes were collected at 16-19 days after fertilization and the pseudo-seeds were removed from the spikes. Picked pseudo-seeds were sterilized in 70% alcohol for 1 minute. Then, they were kept in 20% bleach (Domestos, 4.26% sodium hypochlorite) for 15 minutes and rinsed with sterile distilled water at least 3 times. The haploid embryos were carefully removed from pseudo-seeds under a stereo microscope and cultured on MS medium (Murashige and Skoog, 1962) containing 5% sugar and, 0.7% agar and were kept in the dark condition at 22±0.5°C for 1-2 weeks. Germinated embryos were

transferred in ½ MS media containing 3% sugar and 0.7% agar and kept for 2 weeks under the photoperiod of 16/8 (light/dark) at 22±0.5°C. When plants growing from haploid embryos reached to 5-6 cm, they were transferred to pots containing peat and vermiculite (3:1). Haploid plants with 2-3 tillers were removed without damaging their roots and colchicine doubling solution [colchicine (0.45 g/l) + DMSO (20 ml/l) + GA3 (100 mg/l) + Tween

80 (0.3 ml/l), pH = 5.5] was applied (Niu et al., 2014). These treated plants were rinsed under tap water overnight, and then they were planted to pots again and transferred to the climate cabinet at 18 ±0.5°C and 75% humidity.

Chromosome observations were performed by modifying the method as described by Arslan et al. (2012). First, root tips of haploid plants were kept in alpha-monobromonaftalin solution for 3 hours and then fixed in glacial acetic acid for 30 minutes at room temperature. The fixed root tips were hydrolysed in 1N HCL for 12 minutes at 60°C. Then, root tips were left to be stained for 1 - 1.5 hours in 1 % aceto-orsein solution.

The data of individual factors were given with standard error and interaction between wheat and maize genotypes was measured by the chi-squared test (χ2) for developed pseudo-seeds, embryo, and plantlets.

Results and Discussion

The successful application of wide hybridization in wheat breeding programs is dependent on ability to form pseudo-seeds, embryo and plant regeneration of the genotypes. In this study, a total of 2730 pseudo-seeds were obtained from pollinated 150 wheat spikes, and 53 of them (1.94%) generated haploid embryos. Only 8 (0.29%) of the haploid embryos were regenerated, and 4 (0.14%) of them were improved seed set.

The effect of wheat F1 hybrids on developed

pseudo-seeds, embryo formation, and plant regeneration indicated differences (Table 2). The highest number of developed pseudo-seeds, embryo formation and plant regeneration were recorded in DH6 × Altay 2000, DH20 × Kate A-1 and DH21 × Kate A-1, respectively. High numbers of developed pseudo-seeds and embryo-forming wheat F1

hybrids did not produce a high number of green plants. It is still a controversial issue of whether haploid embryo formation was affected by wheat and maize genotypes. Some researchers observed embryo percentage affected only by maize cultivars, not wheat genotypes, whereas numerous other studies proved wheat genotypes distinctly affect the production of embryos per florets (Filomena Martins-Lopes et al., 2001; Niroula and Thapa, 2009, Xynias et al., 2014).

Table 1. Pedigree of F1 hybrids used as donor plant and their anther culture response.

Pedigree Callus induction Plant regeneration Fertile plants Gained seeds

DH6 × Altay 2000 22 2 0 0 DH18 × Altay 2000 75 0 0 0 DH18 × Kate A-1 111 3 3 205 DH19 × Altay 2000 152 17 5 351 DH20 × Kate A-1 13 0 0 0 DH21 × Kate A-1 119 16 4 458

Avcı and Kutlu / Turkish Journal of Agriculture - Food Science and Technology, 8(7): 1548-1552, 2020

Table 2. Effect of wheat F1 hybrids on the yield of developed pseudo-seeds, embryos and plants

Wheat F1 hybrids Developed pseudo-seeds (n) Embryos Plants

%* % DH6 × Altay 2000 117 2.9 0.4 DH18 × Altay 2000 85 0.6 0.4 DH18 × Kate A-1 74 0.7 0.0 DH19 × Altay 2000 108 1.7 0.5 DH20 × Kate A-1 90 5.9 0.3 DH21 × Kate A-1 72 2.5 0.7 Standard error 4.3 1.4 0.4

*Percentage values were calculated using the following formula: (Embryo or Plant numbers/Developed pseudo seeds) × 100 Table 3. Effect of inducers on the yield of developed pseudo seeds, embryos and plants

Inducers Developed pseudo seeds (n) Embryos Plants

%* %

Baron 97 2.1 0.2

Challenger 93 3.5 0

Merit 70 1.0 0.3

Pollen mixture of maize 83 3.4 1.2

Orchard-grass 112 - -

Standard error 4.0 1.0 0.7

*Percentage values were calculated using the following formula: (Embryo or Plant numbers/Developed pseudo seeds) × 100 Table 4. The effect of bread wheat F1 hybrids × inducers interaction on developed pseudo seeds (number)

Wheat F1 hybrids

Inducers

Baron Challenger Merit Mixture of maize Orchard-grass

DH6 × Altay 2000 134 113 53 122 161 DH18 × Altay 2000 78 81 47 86 132 DH18 × Kate A-1 75 89 48 71 88 DH19 × Altay 2000 108 76 106 100 150 DH20 × Kate A-1 106 114 96 59 76 DH21 × Kate A-1 82 86 67 60 66

Interaction is significant at P=0.01(df=20) since the calculated χ² =105.6> theoretical χ²= 37.56.

Table 5. The effect of bread wheat F1 hybrids × inducers interaction on embryo formation (%)

Wheat F1 hybrids Inducer

Baron Challenger Merit Pollen mixture of maize

DH6 × Altay 2000 2.2 3.5 0 5.7 DH18 × Altay 2000 1.2 0 0 1.2 DH18 × Kate A-1 0 1.1 2 0 DH19 × Altay 2000 0 3.9 0 4 DH20 × Kate A-1 7.5 7.9 0 6.8 DH21 × Kate A-1 0 3.5 4.5 1.7

Interaction is significant at P=0.01(df=15) since the calculated χ² =33.0> theoretical χ²= 30.5.

In terms of the inducer effect on haploid plant regeneration, wheat F1 hybrids × D. glomerata

hybridization produced a high rate of developed pseudo-seeds (Table 3). However, none of them had haploid embryos after 16-19 days. Zenkteler and Nitzsche (1984) reported that a high rate of embryos was obtained from wheat × D. glomerata hybridization, but the embryos disappeared after ten days. In terms of embryo formation, Challenger and pollen mixtures of sweet maize showed similar results. However, the effect of the pollen mixture of sweet maize on plant regeneration was found to be higher. Zhang et al. (1996) observed that the effect of maize genotypes on embryo formation and plant regeneration was important, and the effect of wheat genotypes was insignificant.

While the highest developed pseudo-seeds were observed in the hybridization of DH6 × Altay 2000 with low anther response and D. glomerata, the lowest value was obtained from DH18 × Altay 2000 and Merit hybrid (Table 4). The highest value (7.9 %) for embryo formation occurred in the DH20 × Kate A-1 with low anther response and Challenger hybrid (Table 5). In terms of plant regeneration from haploid embryos, the effect of interaction was found insignificant (Table 6). Although Challenger induced the high rate of embryo formation in wheat F1 hybrids, its effect was not observed in plant

regeneration. Lefebvre and Devaux (1996) and Cherkaoui et al. (2000) reported that wheat × maize interaction was significant in haploid embryo formation and plant regeneration.

1551

Wheat F1 genotypes Inducer

Baron Merit Pollen mixture of maize

DH6 × Altay 2000 0 0 1.7 DH18 × Altay 2000 1.3 0 0 DH18 × Kate A-1 0 0 0 DH19 × Altay 2000 0 0 2 DH20 × Kate A-1 0 0 1.7 DH21 × Kate A-1 0 1.5 1.7

Interaction is not significant at P=0.01(df=10) since the calculated χ² =13.3< theoretical χ²= 23.2. Table 7. Some characters of regenerated plants in wheat × maize hybrids

Characters

Wheat × sweet maize hybrids DH21×Kate A-1×MP DH6×Altay 2000×MP-1 DH6×Altay 2000×MP-2 DH21×Kate A-1×M Mean Fertile spike (%) 19.3 9.7 40.0 38 26.75

Seed number per fertile tiller 13.5 1.3 10.5 14 9.83

MP and M indicate pollen mixture of sweet maize and cv. Merit, respectively.

Among the wheat genotypes, only plants obtained from DH6 × Altay 2000 with low anther response and DH21 × Kate A-1 with high anther response genotypes have produced seeds (Table 7). The number of fertile spikes ranged from 9.7% in DH6 × Altay 2000 × MP-1 to 40% in DH6 × Altay 2000 × MP-2, and the mean fertile spike was 26.75%. The mean seed number per fertile spike was 9.83 and the lowest value was obtained from DH6 × Altay 2000 × MP-1. In the previous study indicated in Table 1, no plants were obtained from the combination of DH6 × Altay 2000 among these genotypes by the anther culture method (Kutlu et al., 2019). It is a pleasing result to obtain high number of seeds from two plants obtained by the wheat × maize crossbreeding method.

Consequently, the efficiency of D. glomerata and sweet maize genotypes for haploid plant production in some bread wheat F1 hybrids were determined. Even though

there was a high rate of developed pseudo-seeds in the wheat × orchard-grass hybrids, no haploid embryo was found. Thus, effects of different hormone type and times of embryo rescue should be evaluated for haploid embryo production between wheat and orchard grass cross. In wheat × sweet maize hybrids, four green plants in two different wheat F1 hybrids were obtained. Differences in

haploid plant regeneration were observed according to the wheat and maize genotypes. Achieving for haploid embryo formation and regeneration from wheat genotypes with a low anther culture response suggest that wheat × maize wide hybridization will be a good alternative to obtain efficient doubled haploid plant regeneration in bread wheat genotypes with low anther response. In genotypes with good anther response and good maternal haploids, the haploid technique to be used is optional. However, the high rate of albino plants may make it preferable to wide hybridization instead of anther technique. Although the source of the pollen to be used depends on the compatibility with the wheat genotypes to be used as a female parent, the effective one on many genotypes can be chosen. However, as proved in this study, applying maize pollen by mixing will guarantee embryo production.

Acknowledgements

This study was supported by Eskişehir Osmangazi University under grant no: 2018-23D01.

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